Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach

Abstract

Background Endothelial colony forming cells (ECFCs), alone or in combination with mesenchymal stem cells, have been selected as potential therapeutic candidates for critical limb-threatening ischemia (CLTI), mainly for those patients considered as “no-option,” due to their capability to enhance revascularization and perfusion recovery of ischemic tissues. Nevertheless, prior to translating cell therapy to the clinic, biodistribution assays are required by regulatory guidelines to ensure biosafety as well as to discard undesired systemic translocations. Different approaches, from imaging technologies to qPCR-based methods, are currently applied. Methods In the current study, we have optimized a cell-tracking assay based on DiR fluorescent cell labeling and near-infrared detection for in vivo and ex vivo assays. Briefly, an improved protocol for DiR staining was set up, by incubation of ECFCs with 6.67 µM DiR and intensive washing steps prior cell administration. The minimal signal detected for the residual DiR, remaining after these washes, was considered as a baseline signal to estimate cell amounts correlated to the DiR intensity values registered in vivo. Besides, several assays were also performed to determine any potential effect of DiR over ECFCs functionality. Furthermore, the optimized protocol was applied in combination with qPCR amplification of specific human Alu sequences to assess the final distribution of ECFCs after intramuscular or intravenous administration to a murine model of CLTI. Results The optimized DiR labeling protocol indicated that ECFCs administered intramuscularly remained mainly within the hind limb muscle while cells injected intravenously were found in the spleen, liver and lungs. Conclusion Overall, the combination of DiR labeling and qPCR analysis in biodistribution assays constitutes a highly sensitive approach to systemically track cells in vivo. Thereby, human ECFCs administered intramuscularly to CLTI mice remained locally within the ischemic tissues, while intravenously injected cells were found in several organs. Our data corroborate the need to perform biodistribution assays in order to define specific parameters such as the optimal delivery route for ECFCs before their application into the clinic.

Effects of Low-Intensity Pulsed Ultrasound on the Migration and Homing of Human Amnion–Derived Mesenchymal Stem Cells to Ovaries in Rats With Premature Ovarian Insufficiency

“…First, the efficacy of LIPUS may be actually not significant. Second, PKH26 Red Fluorescent Cell Linker Kit used to track and locate the transplanted hAD-MSCs in rats is probably less sensitive than the near-infrared (NIR) dye carbocyanine 1,1-dioctadecyl-3,3,3,3 tetramethylindotricarbocyanine iodide (DiR) 70 or Luciferase 71 for in vivo imaging, which might influence the observation and quantification of the distribution of hAD-MSCs in ovaries and other organs of POI rats. Third, the technique 19 used to quantify the number of hAD-MSCs might have deviation, which is not able to accurately calculate the number of hAD-MSCs in the whole ovaries and other organs limited by current technology.…”